The Geobacillus stearothermophilus splG gene encodes a thermophilic spore photoproduct lyase (splG) which belongs to the family of radical SAM enzymes. The aerobically purified apo-splG forms a homodimer, which contains one [4Fe-4S] cluster per monomer unit after reconstitution to the holo form. Formation of the [4Fe-4S] cluster was proven by quantification of the amount of iron and sulfur per homodimer and by UV and EPR spectroscopy. The UV-spectrum features a characteristic absorbance at 420 nm typical for [4Fe-4S] clusters and the EPR data were found to be identical to those of other proteins containing an [4Fe-4S]+ center. Probing of the activity of the holo-splG with oligonucleotides containing one spore photoproduct (SP) lesion at a defined site proved that the enzyme is able to turn over substrate. In addition to repair we observed cleavage of SAM to generate 5´-deoxyadenosine. In the presence of azaSAM the splG is completely inhibited, which provides direct support for the repair mechanism.